Standardization of therapeutic curare preparations



Patented Feb. 8, 1949 STANDARDIZATION THERAPEUTIC CURARE PREPARATIONS Horace A. Holaday, Highland Park, N. J., assignor,

to E. R. Squibb & Sons, New York, N. Y., a corporation of New York; dedicator to the People of the United States No Drawing. Application December 13, 1945, 7

Serial No. 634,898

11 Claims. 1

This application is a continuation-impart of my application Serial No. 396,871, filed June 6, 1941 (now Patent No. 2,397,417, dated March 26, 1946).

This invention relates to, and has for its object the provision of, stable, physiologically-standardized curare preparations suitable for therapeutic use, and a simple and eiiicient method of producing them.

Curare is a valuable agent for the treatment of dystonic and spastic conditions, and for the prevention of spinal and extremity fracturesin the metrazol (pentamethylenetetrazol) and electroshock therapy of mental conditions; but its use has been limited because stable, physiologicallystandardized preparations thereof suitable for therapeutic use-particularly by intravenous injectionhave heretofore been unobtainable. The difficulty of providing curare preparations of constant activity and action is due to the fact that commercial (crude) curare is a variable and physically unstable mixture. Since curares margin of safety (the range of dosage between that giving the desired skeletal-muscle effect and that giving the undesirable toxic paralysis of the muscles of respiration) is small, it is manifestly essential for therapeutic utilitythat the curare prep-- aration be stable and of accurate potency.

In accordance With this invention, stable, physiologically-standardized curare preparations suiti fication treatment removes inert material and,

material having undesirable physiological prop: erties; and the thus-purified curare is highly active, and forms a stable aqueous solution (i. e.,

- the solution remains clear).

Preferably, the crude curare is first extracted with cold water, the water evaporated, and the completely dry residue subjected to extraction with a dry, volatile, organic curare-solvent; the preliminary water extraction serving to prevent extraction by the organic solvent of water-insoluble impurities. Preferably, also, the dry, volatile, organic curare-solvent is' an anhydrous lower aliphatic, monohydric alcohol, and the extraction is eifected at an elevated temperature. The utilizable solvents include, inter'alia, substantially fill A. Standardization of the curare solution A sample of a solution of purified curare, obtained (for example) by the procedure described under the heading A. Purification of crude curare in my Patent No. 2,397,417, is assayed to determine its curare-eifect activity in terms of a suitable standard material. For this purpose, the following specific, rapid, and quantitative method of biological assay has been devised.

The method is based on the observation that injection of a curare preparation into a readily accessible vein of a mammal (e. g., the ear vein of a rabbit) will produce a temporary paralysis of the skeletal muscles (e. g., the muscles of the head and neck and extremities of the rabbit) without paralyzing the respiratory muscles, and if the dose and rate of injection is properly controlled these muscles will reach a degree of flaccidity after injection has been continued for a certain time (e. g., 4-lminutes in the case of rabbits) which just prevents the mammal from using them while suitably positioned (e. g., prevents the'rabb-it from raising its head when tied belly-down on an animal board).

When a rabbit is tied belly-down on an animal board it normally holds its head up. ,The curare preparation is injected into the "ear vein at a slow, constant rate (e. g., 0.10 co. every 15 seconds) until the rabbits head begins to drop; then the rate is'reduced(e. g., to 0.05 co. every 15 seconds) until the rabbits head rests on the board and. the rabbit is unable to raise its head even when stimulated. The paralysis is of short duration (8-15 minutes).

Since the doses of curare required by different.

rabbits vary as much as 150%, but the doseof curare required by eachrabbit for tests on successive days remains constant, the rabbits are crossed-over to obtain a reliable assay. Thus, the curare preparation to be assayed is tested one day on at least 8 rabbits, and an equal numbar are subjected to the test using a solution of the reference standard. On the following day, the rabbits are crossed-over, i. e., those which were injected with the standard are now injected with the preparation to be assayed, and vice versa.

The activity ratio of the sample to be assayed to the reference standard is calculated directly from the corresponding volumes of test sample and reference standard required in each rabbit. The probable error of the average of 16 ratios is usually less than 2%. The accuracy of this biological standardization has received ample clinical confirmation.

Using the foregoing cross-over rabbit-headdrop test, the activity of the reference standard curare may be related to that of a definite chemi cal compound, e. g., quinine methochloride, or (preferably) d-tubocurarine chloride. Thus, the dried crude curare used as a reference standard was found to have 5.26 times the activity of quinine methochoride and .15 the-activity of=, dtubocurarine chloride (pentahydratel); and arunit of activity was considered to be the amount of activity in the aqueous extract .of lzmg. of this reference standard curare.

Preferably, the activity of the curare preparation to be assayed is adjusted to approximately 2 units per cc. (on the basis of a preliminary test with standardized rabbits), so that volumes,

activity and injection-interval are nearly the same for both the preparation to Joe assayed and the ref rence standard. ,By standardized .rab-

bits is meant rabbits which have been'in-jected with the reference standard to determine their respective head-drop doses. Preferably also, each rabbit employed in the assay of this invention is subjectedto at least two priming doses before use (it having been found that, with the same curare preparation-and rabbit, the first and second head-drop doses differ significantly from the third and subsequent doses, whiohare substantially constant).

Although the rabbit'is the animal of choice for the curare assay of this invention (because of the sharpness of the end point, and because the'margi-nal ear vein of the rabbit readily permits of frequent injections), satisfactory assays can be carried out in any other mammalian laboratorytest animal (which can be intravenously injected), including the mouse, rat, dog, cat, monkey. Any sign of relaxation of specificmuscle groups may be chosen as the end-point, for example, ptosis in the monkey ,or human, or relaxation of the jaw in dogs. "For the reasons given hereinbefore in connection with the rabbit assay, it is necessary to make a comparison of the test sample with the standard preparation in each mammal pr ra ly by the cross-over patternue t difiere eesinbedy wei ht of. and munitwe g t dosage for, the different species, approe pr justm o n entration and rate mus b In dehe followin details of assaysin other mammals further illustrate this invention:

i areh d y he tailexte ded throu ene h d st ppe in a g ass cylin er, and inje e with rare prepa a ns (adjust d to about 0.64 unit/cc.) via the tail vein at the rate of 0.005 cc./1 seconds (by use Of. a micrometer syrin o oth d i e u ta le for slow injecon h i jec ion be ng continued until the mouses head falls heavily when raised with a probe. The cross-over pattern is followed.

('2') Dogs are tied loosely .on their sides (on a table) and injected with the curare preparations (adjusted to about 2 units/cc.) via the cephalic or the saphenous .vein at the rate of 3 cc. per minute, the injection being continued until the lateral muscles of the neck and the muscles of the jaw are paralyzed. At that time, the dogs head drops quickly to the table when released from a position of late a ro ation, n h te t c ck a ply as the head strikes the table. The cross-over pattern is followed.

The assay of this invention may be used for the standardization of solutions of the compound d-tubocurarine chloride, as well as solutions of such similarly-acting compounds derived from crude curare as -d-chondocuri ne :dimethochloride and d-tubocurarine dimethyl ether iodide, all such. compounds being comprehended herein (along with curare) by the term curare-drug.

B. Production of a therapeutic cumre preparation The purified and standardized curare solution is diluted with the volume of a saline-diluent solution containing a preservative (preferably chlorbutanol) required to produce a physiological saline preparation of the desired activity. For example, the sodium chloride and chlorbutanol contents of the diluent are adjusted so that the finished preparation will contain 0.5% chlorbutanol and about 0.5% sodium chloride; and the activity is made equivalent to that of an aqueous solution containing 20 mg./cc. of the reference standard curare. The resulting preparation is filtered through a stone filter candle and tested for sterility, and is then ready for therapeutic use. It is a sterile, stable, physiologically-standardized aqueous solution of curare. Since the preparation (usually) contains less than 0.4 (or even 0.33) mg. solids per unit of activity (as herein defined) compared with about 1.0 (or even 0.67) mg. solids per unit of activity in the crude drug, the purified cura-re is over more active than the crude drug. The therapeutic utility of this preparation has beenampl-y confirmed by clinical tests.

The invention may be variously otherwise embodied within the scope of the appended claims.

I claim:

1. In the production of a physiologically-standardize'd curare-drug preparation, assaying the ,curare-drug by determining the relative volumes of aqueous solutions of the test sample and of a reference standardrequired to produce the same temporary paralysis of muscles of, a mammalian laboratory-tested animal when slowly injected intravenously into the same animal under the same conditions at different times, the animal being permitted to recover from one injection before being subjected to the other injection, and making up a preparation of the desired activity from the assayed curare-drug in accordance with the assay results.

2, In the production of a physiologically-standardized curare preparation, assaying the curare by determining the relative volumes of aqueous solutions of the test curare and of a reference standard curare required to produce the same temporary paralysis of muscles of a mammalian laboratory-test animal when slowly injected intravenously into the same-animal under the same conditions at different times, the animal being permitted to recover from one injection before being subjected to the other injection, and making up a preparation .of the desired activity from the assayed curare in accordance with the assay results.

3. In the production of a physiologically-standardized curare preparation, assaying the curare by determining the relative volumes of aqueous solutions of the test curare and of a reference standard curare required to produce the same temporary paralysis of. a rabbits neck muscles when slowly injected into an ear vein of the same rabbit under the same conditions at different times, the rabbit being, permitted to recover from one injection before being subjected to the other injection, and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

4. In the production of a physiologically-standardized curare preparation, assaying the curare by determining the relative volumes of aqueous solutions of the test curare and of a reference standard curare required to produce the same temporary paralysis of a dog's neck and jaw muscles when slowly injected intravenously into the same dog under the same conditions at difierent times, the dog being permitted to recover from one injection before being subjected to the other injection, and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

5. In the production of a physiologically-standardized curare preparation, assaying the curare by determining the relative volumes of aqueous solutions of the test curare and of a reference standard curare required to produce the same temporary paralysis of a mouses neck muscles when slowly injected intravenously into the same mouse under the same conditions at different times, the mouse being permitted to recover from one injection before being subjected to the other injection, and making up a preparation of the desired activity from the assayed curare in accordance with the assay results,

6. In the production of a physiologically-standardized curare-drug preparation, assaying the curare drug by (I) determining the volumes of an aqueous solution of the test sample and of an aqueous solution of the reference standard required to produce in mammalian laboratory-test animals A and B, respectively, the same temporary paralysis of the skeletal muscles when slowly injected intravenously under the same conditions, and (II) after the animals have recovered from said injections, repeating the determination with the animals A and B crossed-over, and making up a preparation of the desired activity from the assayed curare-drug in accordance with the assay results.

'7. In the production of a physiologically-standardized curare preparation, assaying the curare by (I) determining the volumes of an aqueous solution of the test curare and of an aqueous solution of the reference standard curare required to produce in mammalian laboratory-test animals A and B, respectively, the same temporary paralysis of the skeletal muscles when slow- 1y injected intravenously under the same conditions, and (II), after the animals have recovered from said injections, repeating the determination with the animals A and B crossed-over and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

8. In the production of a physiologically-standardized curare preparation, assaying the curare by (I) determining the volumes of an aqueous solution of the test curare and of an aqueous solution of the reference standard curare required to produce in rabbits A and B, respectively, the same temporary paralysis of the neck muscles when slowly injected intravenously under the same conditions, and (II), after the rabbits have recovered from said injections, repeating the determination with the rabbits A and B crossed-over,and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

9. In the production of a physiologically-standardized curare. preparation, assaying the curare by (I) determining the volumes of an aqueous solution of the test curare and of an aqueous solution of the reference standard curare required to produce in groups A and B, respectively, of rabbits the same temporary paralysis of the neck muscles when slowly injected intravenously under the same conditions, and (II), after the rabbits have recovered from said injections, repeating the determination with the groups A and B crossed-over, and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

10. In the production of a physiologicallystandardized curare preparation, assaying the curare by (I) determining the volumes of an aqueous solution. of the test curare and of an aqueous solution of the reference standard curare required to produce in dogs A and B, respectively, the same temporary paralysis of the neck and jaw muscles when slowly injected intravenously under the same conditions, and (II), after the dogs have recovered from said injections, repeating the determination with the dogs A and B crossed-over, and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

11. In the production of a physiologicallystandardized curare preparation, assaying the curare by (I) determining the volumes of an aqueous solution of the test curare and of an aqueous solution of the reference standard curare required to produce in mice A and B, respectively,

' the same temporary paralysis of the neck muscles when slowly injected intravenously under the same conditions, and (II), after the mice have recovered from said injections, repeating the determination with the mice A and B crossed-over, and making up a preparation of the desired activity from the assayed curare in accordance with the assay results.

HORACE A. HOLADAY.

REFERENCES CITED The following references are of record in the file of this patent:

Pharmacopoeia of U. S., 11th revision, Mack Printing Co., 1941, page 148.

Munch, Bioassays, Williams and Wilkins 00.,

. 1931, pages 158-163. 

